1. Field of the Invention
This invention relates to a process for liquefying starch. More particularly it relates to an appropriate process for liquefying starch by using a thermostable .alpha.-amylase which has an optimum working pH value in an acidic region and a low calcium requirement.
2. Description of the Prior Art
In order to prepare sweeteners such as glucose or isomerized sugars, starch is hydrolyzed with an .alpha.-amylase into dextrins which are further decomposed and/or isomerized with other enzymes such as glucoamylase and glucose isomerase. Furthermore .alpha.-amylases have been widely employed to desize fabrics. Recently mesophilic enzymes have been replaced by thermostable .alpha.-amylases. Thermostable enzymes are generally more stable to heating and pH changes than mesophilic enzymes so that the former are very useful in an enzymatic industry.
Conventional .alpha.-amylases are limited to those originating from aerobic bacterial (cf. Shimamura et al., Japanese Patent Publication No. 12946/1971, and L. L. Campbell et al., J. Biol. Chem., 236, 2952 (1961). Among these .alpha.-amylases, those originating from Bacillus subtilis and Bacillus licheniformis have been commercially produced and used in processing starch and desizing fabrics. These known .alpha.-amylases can not exhibit the thermostability with the protein of themselves until a calcium ion is added thereto. They require a calcium concentration of usually several mM to 20 mM (cf. Hattori, Japanese Patent Laid-Open No. 44652/1976 and No. 44690/1976) and at least 1 mM (cf. Saito, Japanese Patent Laid-Open No. 35083/1973). Thus the thermostability of conventional .alpha.-amylases would be significantly decreased at a calcium concentration of 1 mM or below or in the absence of calcium, as shown by the thermostability of an .alpha.-amylase originating from B. licheniformis (cf. Japanese Patent Laid-Open No. 12946/1971 and No. 35083/1973). Therefore a very low calcium concentration (100 .mu.M or below) corresponding to that in tap water would result in inactivation of the enzyme during the reaction so that the expensive enzyme would be wasted. Thus several mM of a soluble calcium salt such as calcium chloride is usually added for the reaction. However in the case of the preparation of final products such as glucose or isomerized sugars by processing starch, it is necessary to remove the calcium after the reaction, which would significantly increase the work load during the desalting step with an ion exchange resin.
In general, .alpha.-amylase has a optimum pH value of 6 or above and few .alpha.-amylases would exhibit a high activity in an acidic region. For example, an .alpha.-amylase originating from B. licheniformis has been known as an acidic .alpha.-amylase (cf. Tanaka et al., Japanese Patent Laid-Open No. 151970/1977 and Saito et al., Japanese Patent Laid-Open No. 35083/1973). In a process of liquefying starch, the starch is generally suspended at a concentration of 10 to 30% and then heated. During this step, organic acids contained in the starch material reduce the pH value of the suspension below 5, or frequently below 4. Therefore the suspension is neutralized to a pH value of 6 to 7 by adding slaked lime or calcium carbonate prior to the treatment with .alpha.-amylase (cf. Ueno, Japanese Patent Laid-Open No. 19049/1974 and Nakajima et al., Japanese Patent Laid-Open No. 55857/1974).
As described above, glucoamylase is used to hydrolyze the dextrin solution obtained by the above liquefaction into glucose. Since conventional glucoamylase has an optimum pH of 4.5 to 5.0, and acid is further added to acidify the dextrin solution after the neutralization during the liquefying step followed by the hydrolysis. Further neutralization is necessary to separate the produced glucose. Therefore it is necessary to add a neutralizing agent for pH adjustment other than a calcium salt as a protective factor when a conventional thermostable .alpha.-amylase is employed, resulting in not only consumption of a reagent but also an additional load in the subsequent desalting step with an ion exchanger. The reaction around neutrality would further bring about the following disadvantages. That is, when the reaction is carried out by heating to a temperature of 80.degree. C. or above, the reduced terminal of a starch molecule would be isomerized, thus causing a decrease in the yield of glucose in saccharification with glucoamylase. In addition, starch would be gelatinized to give a very viscous gel which can be hardly mixed when heated for liquefaction. Under an acidic condition (pH 4), however, the viscosity of the starch gel would fall to approximately 50% of that in a neutral state (pH 6) so that liquefaction in an acidic state is relatively easy.
Up to now, there have been reported no .alpha.-amylases available under a condition wherein these requirements are satisfied, i.e. having a very low calcium requirement and a high thermostability and exhibiting a high activity in an acidic region.